1. Field of the Invention
The invention relates to the cloning of DNA sequences carrying genes encoding mechanisms of resistance to phages in bacteria, in particular lactic acid bacteria.
2. Description of the Related Art
Some bacteria responsible for fermentations are very sensitive to attack by bacteriophages, which, given the importance of lactic acid fermentations in the food industry, presents a major economic problem. Numerous attempts have consequently been made for several years, in order to offer a solution to this problem.
It was proposed, in a first instance, to use in the fermentation, natural mutants or mutants obtained by mutagenesis, and selected for their resistance to bacteriophages. This approach is however considerably limited by the fact that, in most cases, these strains possess other characteristics which are unfavourable for their use, and which are, for example, a slow growth or alternatively the production of metabolites which alter the organoleptic qualities of the finished product. In addition, a reversion to the wild phenotype, and therefore a loss of resistance, or the appearance of sensitivity to other types of phages is frequently observed under the conditions of industrial use of these strains [GASSON and DAVIES, in Advances in the Microbiology and Biochemistry of Cheese and Fermented Milk, Davies F. L. & Law B. eds, 127-151, Elsevier Applied Science Publishers (1984)]. The consequence of this is that a very small number of mutant strains is in effect used industrially.
However, the study of these strains has made it possible to reveal the existence of several different mechanisms of resistance to phages.
Three principal mechanisms of resistance to phages have thus been described in the resistant strain Lactococcus lactis ME2 [KLAENHAMMER, J. Dairy Sci., 72, 3429-3443, (1989)].
In one of them, called "mechanism of interference with adsorption", the product of the resistance gene delays the absorption of the phage on the bacterium.
A second mechanism called "mechanism of restriction-modification" (R/M), calls into play a restriction endonuclease which degrades the phage DNA as soon as it enters the bacterium. However, this endonuclease is linked to a methylase which has the same specificity; it follows that some phage DNA modified by the methylase may escape the action of the endonuclease and give rise to infectious particles. Several R/M systems of differing specificities have been described; these systems have a variable efficiency: the frequency of appearance of viral particles containing a modified DNA is between 10.sup.-1 and 10.sup.-8. Under the conditions of industrial culture when during an infection very many viral particles appear, the efficiency of this system is therefore inadequate. However, when several R/M systems are present in the same bacterium, the protection offered appears more efficient [JOSEPHSEN and KLAENHAMMER, Plasmid, 23, 71-75 (1989)].
The genes encoding the R/M type mechanisms known so far are all located in the plasmid.
The third type of defense mechanism which has been described is called "mechanism of abortive infection" (Abi). In the bacterial strains which possess this type of mechanism, the adsorption of phages is normal, but the multiplication of phages to give rise to new viral particles occurs only in a few cells. Moreover, the phages resulting therefrom are not capable of producing a complete lytic cycle when they infect a new cell.
All the Abi type mechanisms described so far are encoded by genes carried by plasmids [MCKAY et al., Appl. Environ. Microbiol., 47, 68-74 (1984)]; [KLAENHAMMER and SANOZKY, J. Gen. Microbiol. 131, 1531-1541 (1985)]; [GAUTIER and CHOPIN, Appl. Environ. Microbiol. 53, 923-927 (1987)]; [DALY and FITZGERALD, in Streptococcal Genetics, Ferretti J. & Curtiss R. eds, III, 259-268 ASM, Washington D.C. (1987)]; [LAIBLE et al., J. Dairy Sci. 70., 2211-2219 (1987)]; [MURPHY et al., Appl. Environ. Microbiol. 54, 777-783 (1988)]; [JARVIS, Appl. Environ. Microbiol. 53, 777-783 (1988)]; [FROSETH et al., J. Dairy Sci. 71, 275-284 (1988)].
The gene encoding an Abi-type resistance mechanism, called Hsp, which is carried by the plasmid pTR2030 has been cloned and sequenced [HILL et al., Appl. Environ. Microbiol., 56, 2255-2258, (1990)].
The modes of action of the Abi-type resistance mechanisms have not yet been elucidated; it appears however that abortive infection may result from several systems having different mechanisms of action, active on different phages and, at different levels, on viral proliferation. It has for example been shown that the plasmid pTR2030 is more active on the phages belonging to the so-called group of "small phages with isometric heads" than on those belonging to the group of "big phages with isometric heads" or to the group of phages "with elongated heads".
Similar observations concerning a specific resistance to attack by a group of phages have been made for other plasmids carrying genes encoding Abi-type resistance mechanisms [DALY and FITZGERALD, (1987); FROSETH et al., (1988); MURPHY et al., (1988) (publications mentioned above); STEELE and MCKAY, Plasmid, 22, 32-43 (1989)].
However, other plasmids, for example the plasmid pIL105 [GAUTIER and CHOPIN, App. Environ. Microbiol., 53, 923-927 (1987)], and pAJ1106 [JARVIS, App. Environ. Microbiol. 54., 77-783 (1988)] encode Abi resistance mechanisms which have a different specificity from the preceding plasmids and are effective both on "isometric" phages and on phages "with elongated heads".
Other observations also show that several Abi-type resistance systems exist; hybridisation experiments between different Abi plasmids have suggested that different loci were involved [STEELE et al., Appl. Environ. Microbiol., 55, 2410-2413 (1989)]. It has also been observed that the resistance to phages was increased when several DNA sequences encoding Abi mechanisms were linked.
The use of plasmids encoding mechanisms for resistance to phages to transfer this resistance to the strains used in industry has been proposed. For example, bacterial strains containing the autotransferable plasmid pTR2030 have been successfully used in industrial fermentations, and they exhibited satisfactory resistance to bacteriophage attack, which shows the real usefulness of Abi-type resistance genes.
However, the plasmid pTR2030 like most phage resistance plasmids described in the prior art and whose use has been suggested, is a "natural" plasmid present in resistant strains and transmitted to other strains by bacterial conjugation. The use of such plasmids has certain limits; these plasmids should be transferable or they should be capable of being linked to a plasmid vector; they should also be stable inside the host bacterium and should not contain genes which modify its phenotype in an unfavourable manner; furthermore, in order to be able to combine in the same bacterium-various mechanisms of resistance to phages, the plasmids concerned should be mutually compatible. Finally, this technique does not enable the transfer of resistance genes which may be carried, not by the plasmid DNA, but by chromosomal DNA. It is therefore particularly desirable to clone the genes responsible for these resistance mechanisms so as to be able to insert them at will inside suitable recombinant vectors which are mutually compatible and which are compatible with the host bacterium whose transformation is desired.